Exploration of KIR genes and hematological-related diseases in Chinese Han population

The function of natural killer (NK) cells has previously been implicated in hematopoietic-related diseases. Killer immunoglobulin-like receptors (KIR) play an important role in NK cells after hematopoietic stem cell transplantation. To explore the immunogenetic predisposition of hematological-related diseases, herein, a multi-center retrospective study in China was conducted, analyzing and comparing 2519 patients with hematopathy (mainly, acute lymphoblastic leukemia, acute myeloid leukemia, aplastic anemia, and myelodysplastic syndrome) to 18,108 individuals without known pathology. Genotyping was performed by polymerase chain reaction with specific sequence primers (PCR-SSP). As a result, we discovered four genes including KIR2DL5 (OR: 0.74, 95% CI 0.59–0.93; Pc = 0.0405), 2DS1 (OR: 0.74, 95% CI 0.59–0.93; Pc = 0.0405), 2DS3 (OR: 0.58, 95% CI 0.41–0.81; Pc = 0.0180), and 3DS1 (OR: 0.74, 95% CI 0.58–0.94; Pc = 0.0405) to be protective factors that significantly reduce the risk of aplastic anemia. Our findings offer new approaches to immunotherapy for hematological-related diseases. As these therapies mature, they are promising to be used alone or in combination with current treatments to help to make blood disorders a manageable disease.

The association between KIR genes and hematopoietic-related diseases. Further analysis of data from various categories of hematology revealed the differences in the KIR genes between patients with hematopoietic-related diseases and controls. Figure 1 shows that the susceptibility of patients with AA and ALL to the KIR genes differed considerably from that of individuals without known pathology (2DL5, 2DS1, 2DS3, and 3DS1 for AA) (Pc < 0.05), and no equivalent involvement appears to be identified in AML and MDS. Other Table 1. Characteristics of KIR gene frequency in the study population. OR: odds ratio, which describes the odds of cases being KIR genes carriers to the odds of controls being KIR genes carriers. www.nature.com/scientificreports/ KIR genes including 2DL1-2DL3, 3DL1-3DL3, 2DS2, 2DS4, and two pseudogenes (2DP1 and 3DP1) displayed no significant association with AML and MDS. Furthermore, during the examination of disease-associated genetic polymorphisms, an inhibitory KIR gene and three activating KIR genes were discovered to be protective genes for AA: the former was 2DL5 (OR: 0.74, 95% CI 0.59-0.93; Pc = 0.0405); the latter consisted of 2DS1 (OR: 0.74, 95% CI 0.59-0.93; Pc = 0.0405), 2DS3 (OR: 0.58, 95% CI 0.41-0.81; Pc = 0.0180), and 3DS1 (OR: 0.74, 95% CI 0.58-0.94; Pc = 0.0405) (Fig. 2). On the other hand, it appears that no susceptible KIR genotypes have been associated to AML and MDS. Moreover, we performed further analysis by grouping gender and age according to different disease types, but found that there was no statistically significant difference in the KIR gene frequencies between the different gender groups as well as between the various age groups (Tables S1-S8).

Discussion
Around the hypothesis that "similar immunogenetic predisposition exists in all or specific subgroups of hematology", we conducted a retrospective study in a large-scale multi-center Chinese population to analyze the susceptibility of KIR genes in ALL, AML, AA, and MDS. As a result, our study successfully identified the KIR protective genes associated with AA (2DL5, 2DS1, 2DS3, and 3DS1). These findings could contribute reliable evidence for a potential association between KIR genes and hematopoietic-related diseases. This provides more in-depth basic research for clinical treatment.
As a multi-gene family, KIR gene diversity is achieved through differences in gene content and genetic polymorphism. In this study, we found that inhibitory genes (i.e., 2DL1, 2DL3, 3DL2, and 3DL3), activating and inhibiting potential genes (i.e., 2DL4), and pseudogenes (2DP1 and 3DP1) of KIR were common in all patients and controls. On the contrary, the frequency of activating KIR genes (excepting 2DS4), was lower than that of all inhibitory KIR genes except 2DL2 and 2DL5. Consistently, in a study based on the Han population in northern China, the frequencies of KIR3DL3, 3DL2, and 2DL4 reached 100% in all subjects (including 50 leukemia patients and 60 healthy donors) 19 . Strong linkage disequilibrium between KIR genes may contribute to this phenomenon,   21 . Another study suggested that MDS patients had higher frequencies of 2DS5 and lower frequencies of 2DL3 when compared to healthy individuals 22 . Hence, to eliminate the effect of multiple diseases confounding the outcomes, we subdivided the blood disorders in the next analysis into four types: AML, ALL, AA, and MDS, with MDS progressing to AML. This enables precise and comprehensive relationships between genetic variations in KIR gene cluster and risk of hematopoietic related diseases.
In view of the various immunogenetic factors, KIR genes may play an important role in enhancing and/or reducing the immune response [23][24][25][26] . Dou et al. investigated KIR2DL1 in AML patients compared with donors (17.6% vs. 75%, Pc = 0.03) 27 . Our inconsistent results with this report require further advanced research to follow up. On the other hand, by comparing the susceptibility to KIR genes in AA and MDS patients with controls, 2DL5, 2DS1, 2DS3, and 3DS1 were found to be protective factors for AA. In this regard, no association was found between the KIR genotype and MDS. Based on this, Serio et al. reported a decreased frequency of the inhibitory KIR2DL3 gene for AA but no difference in the frequency of the KIR genotype for MDS 21 , which is consistent with our findings. Moreover, it has been shown that KIR genotypes and cytokines, as well as cytokine receptor gene SNPs, may play a role in the important susceptibility to excessive and/or reduced immune response 28,29 . For instance, expression of the KIR3DS1 ligand HLA-F is selectively lost on KIR-L (-) primitive hematopoietic stem cells derived from 6pLOH (+) induced pluripotent stem cells in KIR3DS1 (+) patients 30 . And Zeng et al. found an association between toll-like receptor genes in bone marrow CD4+ cells and overexpression of KIR genes in bone marrow CD8+ cells in AA patients 31 . Overall, following the above studies, the relationship between susceptibility genes and the severity and response to AA demands further confirmation.
With several limitations of previous research, our study is the first large-scale multi-center study on KIR in China. The purpose of the study was to explore the frequency distribution and susceptibility of KIR genes among AML, ALL, AA, and MDS patients and individuals without known pathology. Although some of our findings appear inconsistent with previous studies, possible explanations for the discrepancy lie in the differences in cohort size, disease classification, and significant ethnic and regional distribution bias. Besides, despite the diagnostic information of some patients being missing or unclear, a relatively large sample size may attenuate the impact of partial information loss. DNA extraction. Genomic DNA was extracted from peripheral blood using the QIARamp Blood Kit (Qiagen, Hilden, Germany). The purified DNA was dissolved in 0.5 mL of DNA hydration solution and stored at -80℃ for future use. KIR genotyping. The presence or absence of the KIR genes was determined using the Dynal KIR Genotyping kit (Invitrogen Corp, Carlsbad, CA, USA). Genomic DNA was amplified by polymerase chain reaction with specific sequence primers (PCR-SSP) and mixed with 31 μL (50 ng/μL) of genomic DNA sample and reaction buffer, 2.3 μL Taq DNA polymerase (5 U/μL), and 152 μL of water. PCR amplification began with denaturation at 95 °C for 1 min, followed by 30 cycles of amplification at 94 °C for 20 s, 63 °C for 20 s, and 72 °C for 90 s. The PCR products were analyzed on 2% agarose gel. This method was used to type 14 KIR genes and 2 pseudogenes: 2DL1-2DL5, 2DS1-2DS5, 3DL1-3DL3, 3DS1, 2DP1, and 3DP1. KIR genotype frequencies were estimated by counting the number of positive individuals for a given gene in 2% agarose gel.

Study population.
Statistical analysis. All analyses were performed using R version 4.1.0. The chi-square test was applied to compare the variability of each KIR gene in patients and controls. GraphPad Prism 9 was used to compare the frequency distribution of each KIR gene. Prevalence among patients was counted according to diagnostic information and the strength of association was estimated as ORs with 95% CIs. Then pairwise comparisons were adjusted for multiple tests with Bonferroni's correction. An adjusted Pc < 0.05 indicates a statistical significance.
Ethics approval. This is an observational study. The XYZ Research Ethics Committee of Shanghai Tissuebank Medical Laboratory has confirmed that no ethical approval is required.